Global demand for liquid transportation fuel is projected to strain the ability to meet certain environmentally driven goals, for example, the conservation of oil reserves and limitation of green house gas emissions. Such demand has driven the development of technology which allows utilization of renewable resources to mitigate the depletion of oil reserves and to minimize green house gas emissions. This invention addresses the need for improved processes for the conversion of plant-derived raw materials to a product stream useful as a liquid transportation fuel. Such processes would satisfy both fuel demands and environmental concerns.
Butanol is an important industrial chemical and is useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a food grade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase in the future.
Methods for the chemical synthesis of isobutanol are known such as oxo synthesis, catalytic hydrogenation of carbon monoxide (Ullmann's Encyclopedia of Industrial Chemistry, 6th edition, 2003, Wiley-VCH Verlag GmbH and Co., Weinheim, Germany, Vol. 5, pp. 716-719) and Guerbet condensation of methanol with n-propanol (Carlini, et al., J. Molec. Catal. A: Chem. 220:215-220, 2004). These processes use starting materials derived from petrochemicals, are generally expensive, and are not environmentally friendly. The production of butanol from plant-derived raw materials would minimize green house gas emissions and would represent an advance in the art.
Some fungi desire supplementation of the vitamin biotin, like many wild isolates of the yeast Saccharomyces cerevisiae (Leonian, et al., Science 95:658, 1942; Stolz, et al., J. Biol. Chem. 274:18741-18746, 1999; Hall, et al. Genetics 177:2293-2307, 2007). For a biotin auxotrophic organisms to grow, sufficient amount of biotin is provided exogenously. Alternatively, endogenous biosynthesis of biotin can be accomplished, for example, by directed evolution (Leonian, et al., supra) or complementing the retained biosynthesis pathway of yeast with the missing enzyme activities (Hall, et al., supra).
Another vitamin that is often supplemented to yeast cultures is pantothenic acid (Leonian, et al., supra). However, although Saccharomyces cerevisiae is capable of de novo pantothenic acid biosynthesis, an increase of biosynthesis, especially the enzyme activity in the rate-limiting step of amine oxidase, may be achieved by directed evolution (Leonian, et al., supra) or recombinant DNA technology (White, et al., J. Biol. Chem. 276:10794-10800, 2001) which is typically required to provide sufficient activity to remedy growth, respectively. No further vitamin requirements for aerobic growth of Saccharomyces cerevisiae have been identified (Henry, Appl. Environ. Microbiol. 31:395-398, 1976).
A commercial defined medium without yeast extract and containing only the vitamins inositol, biotin, pantothenic acid, and pyridoxine was used to aerobically produce factor XIII with Saccharomyces cerevisiae (see, e.g., U.S. Pat. No. 6,750,045). A commercial medium for the aerobic production of protein in Saccharomyces cerevisiae supplemented with a vitamin mixture comprising biotin, pantothenic acid, myo-inositol, and pyridoxine is described in U.S. Pat. No. 5,795,771. U.S. Patent Application Publication No. 2005/0112737 reports a chemically defined medium supplemented with the vitamins biotin, inositol, and thiamine to grow a pyruvate-decarboxylase (PDC)-negative Saccharomyces cerevisiae with an exogenous lactate dehydrogenase activity. The non-Crabtree and PDC-KO yeast Kluyveromyces marxianus has been shown to produce lactic acid in shake flask cultivations with complex yeast extract peptone-dextrose medium (YPD) media comprising 10 g/L yeast extract, 20 g/L peptone, additionally glucose, and occasionally agar. No additional specific media requirements are described (see, e.g., U.S. Pat. No. 7,534,597). An example of a non-Crabtree yeast Kluyveromyces marxianus capable of generating biomass when cultured with corn fiber hydrolyzate supplemented with yeast minimal medium and a vitamin cocktail (Kiers, et al., Yeast 14:459-469, 1998) where 5 mg/L of nicotinic acid was added to the fiber hydrolysate is described in U.S. Pat. No. 7,700,332. However, no link of bioprocess performance to specific compounds of the vitamin mixture is made.
Very high gravity (VHG) fermentation with Saccharomyces cerevisiae and corn flour mash for the production of ethanol was optimized through supplementations of Mg2+, glycine, yeast extract, biotin, acetaldehyde, and peptone (Wang, et al., Biotechnol. Lett. 29:233-236, 2007). Ethanol production with brewing yeast was also improved by adding a nitrogen source, ergosterol, and oleic acid to high-gravity worts of 16 to 18% dissolved solids (Casey, et al., Appl. Environ. Microbiol. 48:639-646, 1984). It is also reported that in a high cell density fermentation, feeding biotin in combination with a vitamin mixture containing pantothenic acid, meso-inositol, nicotinic acid, thiamine, pyridoxine, and para-aminobenzoic acid during aerated fed-batch processes improves ethanol production and viability of Saccharomyces cerevisiae (Alfenore, et al., Appl. Microbiol. Biotechnol. 60:67-72, 2002). However, apart from biotin, the composition of the vitamin mixture was not further investigated to link observed performance increases to specific components.
A complex YPD medium consisting of 10 g/L yeast extract, 20 g/L peptone, and variable amounts of glucose was described in examples for culturing the yeasts Kluyveromyces marxianus and Saccharomyces cerevisiae to produce butanol (see, e.g., WO 2010/075504). No specific requirements or analyses of vitamin requirements are mentioned. Examples of butanol production in yeasts Kluyveromyces marxianus and Saccharomyces cerevisiae, some with reduced or completely deleted PDC-activities, were described using 6.7 g/L YNB medium without amino acids and 0.076 g/L histidine with nicotinic acid concentrations of about 0.4 mg/L (see, e.g., WO 2010/051527). An economic comparison of nutrient costs in example fermentations is disclosed in U.S. Patent Application Publication No. 2009/0215137. Synthetic fermentation medium (mineral medium) (SFM) is described for butanol production with a yeast cell using a vitamin mixture according to Verduyn, et al., (Yeast 8:501-517, 1992) with the medium containing 1 mg/L nicotinic acid (see, e.g., WO 2009/103533).
Genetic modification of microorganisms to produce new products frequently comes with changed and/or new nutritional requirements to ensure optimum performance of the biocatalyst. Identifying and optimizing nutritional requirements can be complex and feeding complex multi-vitamin mixtures to address nutritional needs can be costly. Consequently, technical inventions are necessary to address and solve these hurdles.